R binding activity is present in all phases of 1 L methanol-free cultivations. Duplicate 1 L bioprocesses for the same strain cultured previously (Figure 1) were analysed for hA2aR binding activity (pmol mg-1; red circles), DCW (g; white circles) and residual glycerol concentration (g L-1; white triangles) in the absence of methanol induction. hA2aR binding activity was measured in all the phases: glycerol batch phase (I); glycerol fed-batch phase (II) and carbon source starvation phase (III). The residual glycerol concentration was at its highest during phase I, dropping to zero in phase II and for the duration of the cultivation. DCW increased during phases I and II and plateaued during phases III. Measurements were made in triplicate. The error bars represent the SEM.